Natural Killer Cell Functional Responses Research Articles

Progressive accumulation of hyperinflammatory NKG2Dlow NK cells in early childhood severe atopic dermatitis.

Atopic dermatitis (AD) is a chronic inflammatory skin disease that often precedes the development of food allergy, asthma, and allergic rhinitis. The prevailing paradigm holds that a reduced frequency and function of natural killer (NK) cell contributes to AD pathogenesis, yet the underlying mechanisms and contributions of NK cells to allergic comorbidities remain ill-defined. Here, analysis of circulating NK cells in a longitudinal early life cohort of children with AD revealed a progressive accumulation of NK cells with low expression of the activating receptor NKG2D, which was linked to more severe AD and sensitivity to allergens. This was most notable in children co-sensitized to food and aeroallergens, a risk factor for development of asthma. Individual-level longitudinal analysis in a subset of children revealed coincident reduction of NKG2D on NK cells with acquired or persistent sensitization, and this was associated with impaired skin barrier function assessed by transepidermal water loss. Low expression of NKG2D on NK cells was paradoxically associated with depressed cytolytic function but exaggerated release of the proinflammatory cytokine tumor necrosis factor-α. These observations provide important insights into a potential mechanism underlying the development of allergic comorbidity in early life in children with AD, which involves altered NK cell functional responses, and define an endotype of severe AD.

Progressive accumulation of hyperinflammatory NKG2D lowNK cells in early life defines a novel endotype of severe atopic dermatitis

Abstract Atopic dermatitis (AD) is a chronic inflammatory skin disease that often precedes the development of food allergy, asthma, and allergic rhinitis. The prevailing paradigm holds that a reduced frequency and function of natural killer (NK) cell contributes to AD pathogenesis, yet the underlying mechanisms and contributions of NK cells to allergic co-morbidities remain ill-defined. Herein, longitudinal analysis of circulating NK cells in a well-defined early life cohort of children with AD revealed a progressive accumulation of NK cells with low expression of the activating receptor NKG2D that was linked to more severe AD and sensitivity to allergens. This was most notable in children co-sensitized to food and aero allergens, a risk factor for development of asthma. Importantly, low expression of NKG2D on NK cells was paradoxically associated with depressed cytolytic function but exaggerated release of the proinflammatory cytokine TNF-a. These observations provide important new insights into a potential pathophysiological mechanism of atopic march involving altered NK cell functional responses and define a novel endotype of severe AD. 5U19AI70235 (GKKH, JBM, LJM); American Heart Association (DEO); R01AI148080, R01AR073228 (SNW), 5T32GM063483 (SBD)

Harnessing natural killer cells for cancer immunotherapy: dispatching the first responders.

Natural killer (NK) cells have crucial roles in the innate immunosurveillance of cancer and viral infections. They are 'first responders' that can spontaneously recognize abnormal cells in the body, rapidly eliminate them through focused cytotoxicity mechanisms and potently produce pro-inflammatory cytokines and chemokines that recruit and activate other immune cells to initiate an adaptive response. From the initial discovery of the diverse cell surface receptors on NK cells to the characterization of regulatory events that control their function, our understanding of the basic biology of NK cells has improved dramatically in the past three decades. This advanced knowledge has revealed increased mechanistic complexity, which has opened the doors to the development of a plethora of exciting new therapeutics that can effectively manipulate and target NK cell functional responses, particularly in cancer patients. Here, we summarize the basic mechanisms that regulate NK cell biology, review a wide variety of drugs, cytokines and antibodies currently being developed and used to stimulate NK cell responses, and outline evolving NK cell adoptive transfer approaches to treat cancer.

Stage-Specific Requirement for Eomes in Mature NK Cell Homeostasis and Cytotoxicity

Natural killer (NK) cells are cytotoxic innate lymphoid cells (ILCs) that mediate antiviral and antitumor responses and require the transcriptional regulator Eomesodermin (Eomes) for early development. However, the role of Eomes and its molecular program in mature NK cell biology is unclear. To address this, we develop a tamoxifen-inducible, type-1-ILC-specific (Ncr1-targeted) cre mouse and combine this with Eomes-floxed mice. Eomes deletion after normal NK cell ontogeny results in a rapid loss of NK cells (but not ILC1s), with a particularly profound effect on penultimately mature stage III NK cells. Mechanisms responsible for stage III reduction include increased apoptosis and impaired maturation from stage II precursors. Induced Eomes deletion also decreases NK cell cytotoxicity and abrogates in vivo rejection of major histocompatibility complex (MHC)-class-I-deficient cells. However, other NK cell functional responses, and stage IV NK cells, are largely preserved. These data indicate that mature NK cells have distinct Eomes-dependent and -independent stages.

Immunometabolism and natural killer cell responses.

Natural killer (NK) cells are lymphocytes with important roles in innate and adaptive immune responses to tumours and viral infection. However, in certain chronic diseases, including obesity and cancer, NK cell functional responses are impaired. Recently, research has highlighted the importance of NK cell metabolism in facilitating robust NK cell effector functions. This Review describes our current understanding of mouse and human NK cell metabolism and the key signalling pathways that mediate metabolic responses in NK cells. Furthermore, it explores howdefects in metabolism can contribute to the generation of dysfunctional NK cells in chronic disease. Finally, the potential for new therapeutic strategies targeting cellular metabolism isdiscussed.

Srebp-controlled glucose metabolism is essential for NK cell functional responses.

Activated natural killer (NK) cells engage in a robust metabolic response that is required for normal effector function. Using genetic, pharmacological and metabolic analyses, we demonstrated an essential role for Srebp transcription factors in cytokine-induced metabolic reprogramming of NK cells that was independent of their conventional role in the control of lipid synthesis. Srebp was required for elevated glycolysis and oxidative phosphorylation and promoted a distinct metabolic pathway configuration in which glucose was metabolized to cytosolic citrate via the citrate-malate shuttle. Preventing the activation of Srebp or direct inhibition of the citrate-malate shuttle inhibited production of interferon-γ and NK cell cytotoxicity. Thus, Srebp controls glucose metabolism in NK cells, and this Srebp-dependent regulation is critical for NK cell effector function.

Human CD56bright NK Cells Acquire Potent Anti-Leukemia Functionality Following IL-15 Priming

Natural killer (NK) cells are innate lymphoid cells that mediate anti-tumor responses via cytotoxicity and effector cytokine production. Human NK cells are divided into two subsets based on relative expression of CD56 (CD56bright and CD56dim) that classically participate in distinct functions. Cytotoxic CD56dim NK cells respond to tumor targets without prior stimulation, resulting in target cell death and transient secretion of effector cytokines (e.g. IFN-γ). In contrast, immunoregulatory CD56bright NK cells secrete abundant IFN-γ and other cytokines in response to cytokine receptor stimulation, but respond minimally to tumor target-based triggering. As a result of this dichotomy, translational strategies to enhance NK cell function for cancer immunotherapy have focused exclusively on the CD56dim subset.Based upon studies in mouse NK cells, we hypothesized IL-15 priming would enhance CD56bright anti-tumor functionality. Primary human NK cells from healthy donors were purified (>95% CD56+CD3-), cultured overnight in medium alone (control) or medium with 5 ng/mL rhIL-15 (primed), washed, and assayed for anti-tumor responses. IL-15 priming significantly enhanced multiple CD56bright NK cell functional responses to the prototypical AML target cell line K562 (CD107a+: control 20% vs. primed 59%, p<0.001; IFN-γ+: 3% vs. 27%, p<0.001; TNF: 3% vs. 20%, p<0.001), as well as primary AML blasts (N=3 unique AML sample: CD107a+,7% vs. 30%, p<0.001; IFN-γ 2% vs. 14%, p<0.001; TNF: 2% vs. 22%, p<0.001). IL-15-priming of CD56bright NK cells was evident after 1 hour, and peaked after only 6 hours. In addition, flow-sorted IL-15-primed CD56bright NK cells exhibited markedly increased killing of leukemia target cells. Similar results for functional comparisons were observed using primary NK cells from AML patients. Unexpectedly, IL-15-primed CD56bright NK cell anti-leukemia responses significantly exceeded those of IL-15-primed CD56dim NK cells. Multidimensional CyTOF analyses revealed that the maturity status of CD56dim NK cells did not determine the extent to which they could be primed by IL-15.In response to IL-15, we observed selective activation of the PI3K/Akt/mTOR (4.2 fold increase CD56bright NK cells, 1.2 CD56dim NK cells, p<0.001) and Ras/Raf/MEK/ERK (1.9 fold increase CD56bright NK cells, 1.2 CD56dim NK cells, p<0.001) pathways in CD56bright NK cells. The Jak/STAT pathway was strongly activated in both CD56bright and CD56dim subsets. These data suggested a signaling mechanism for preferential CD56bright NK cell priming by IL-15. Indeed, small molecule inhibitors of PI3K/Akt/mTOR and Ras/Raf/MEK/ERK pathways abrogated the anti-tumor responses of IL-15-primed CD56bright NK cells, supporting this idea. Several NK cell effector mechanisms were enhanced in IL-15-primed CD56bright NK cells, likely contributing to their augmented anti-tumor responsiveness. These included increased cytotoxic effector protein levels (granzyme B and perforin), as well as improved conjugate formation with tumor targets. Furthermore, blocking experiments demonstrated that IL-15-primed CD56bright NK cell anti-tumor responses depended on LFA-1, CD2, and NKG2D receptor interactions with target cells. Finally, since IL-15-based therapeutics are being investigated in clinical trials, we tested whether the IL-15 super-agonist complex ALT-803 primes CD56bright NK cells in vivo in cancer patients. There was an increase in leukemia target cell-triggered degranulation (CD107a+ unprimed 7% vs. primed 30%, p<0.001) and cytokine production (IFN-γ+ 6% vs. 31%, p<0.01; TNF+ 2% vs. 20%, p<0.05) by CD56bright NK cells 24 hours post-ALT-803 administration to multiple myeloma patients, compared to unprimed, pre-therapy values.Collectively, these results suggest that CD56bright NK cells play an under-appreciated anti-tumor role in settings of abundant IL-15, such as following lymphodepleting chemotherapy, during preparation for stem cell transplantation, at sites of inflammation, or after exogenous IL-15 administration. Since CD56bright NK cells have different in vivo tissue localization (secondary lymphoid organs), distinct inhibitory, activating, and chemokine receptor expression compared to CD56dim NK cells, and are thought to be the most abundant NK cell subset when considering all human tissues, this study identifies a promising NK cell subset to harness for cancer immunotherapy. DisclosuresNo relevant conflicts of interest to declare.

Natural Killer (NK) Cells’ Response to Epstein - Barr virus Infections and its Influence on the Risk of Developing Post-transplant Lymphoproliferative Disease (PTLD) after Allogeneic Hematopoietic Cell Transplantation: Effect of Donor Killer Ig-like Receptor Genes and Motifs Copy Numbers

Abstract BACKGROUND Uncontrolled reactivation of Epstein-Barr virus (EBV) leading to post-transplant lymphoproliferative disorder (PTLD) is one of the major complications after T-cell depleted HCT. Recovering within weeks after HCT, natural killer (NK) cells are deemed important in the immunopathogenesis of EBV complications. Their role however remains elusive. NK cell responses are regulated by a series of activating and inhibitory cell surface receptors, central to which are the Killer Ig-like Receptors (KIR). Here we hypothesized and tested whether diverse NK cell receptor repertoires can titrate NK cell functional responses to EBV and can potentially modify the risk of developing PTLD. METHODS KIR genotypes, centromeric and telomeric motifs and their variants were determined for 356 allo-HCT donors through next generation sequencing of KIR locus. PBMNCs from KIR typed healthy volunteers were co-cultured with EBV-transformed cells and degranulation and IFNγ producing NK cells were enumerated using multi-parameter flow cytometry. Effect of donor KIR profile on PTLD was tested using competing risks regression statistics. Segregation of NK cell response to EBV across various KIR repertoires was tested by Mann-Whitney U statistics RESULTS At least one copy of Donor tA01 motifs was required for a strong protection against PTLD (p=0.0001, SHR=0.17). The number of EBV induced NK cells increased with increasing tA01 motifs. There was no influence of recipients’ KIR repertoire on the risk of developing PTLD. CONCLUSIONS KIR-regulated NK cells have a profound effect on the risk of PTLD. KIR gene profile based identification of HCT recipients at high risk of PTLD will enable closer monitoring of EBV DNAemia and facilitate prompt therapy.

Copies of Donor Killer Immunoglobulin-like Receptor Genes and Motifs Titrate Natural Killer (NK) Cells' Functional Response to Epstein - Barr Virus Infections and Influence the Risk of Developing Post-Transplant Lymphoproliferative Disease (PTLD) after Allogeneic Hematopoietic Cell Transplantation

BACKGROUND: Recipientsof allogeneic HCT remain vulnerable to a heightened risk of reactivation of otherwise latent viral infections owing to a compromised immune system early after transplantation. Uncontrolled reactivation of Epstein-Barr virus (EBV) leading to post-transplant lymphoproliferative disorder (PTLD) is one of such major complications after T-cell depleted HCT. Recovering within weeks after transplantation and being first in line of defense against viral infections, natural killer (NK) cells are deemed important in the immunopathogenesis of EBV complications. Their role however remains elusive. NK cell responses are regulated by a series of activating and inhibitory cell surface receptors, central to which are the Killer Immunoglobulin-like Receptors (KIR). Through these receptors NK cells discriminate healthy cells from 'altered' self-cells by scaling the perturbations in HLA expression after viral transformation of the target cell. Here, we set out to determine whether and how KIR gene and motifs' content of HCT donors and/or recipients influences the development of PTLD after allo-HCT.STUDY DESIGN:Hypothesizing that diverse NK cell receptor repertoires can titrate NK cell functional responses to EBV infections/reactivation and can potentially modify the risk of developing PTLD, we determined the KIR gene repertoires of 356 HLA-matched donor-recipient pairs of first allo-HCT and 50 healthy donors through Next Generation Sequencing of the KIR locus on the Illumina MiSeq platform. Based on the presence/absence and number of copies of individual genes, the KIR genotypes were determined and classified into four common centromeric (cA01, cB01, cB02 and cB03) and two telomeric (tA01 and tB01) motifs along with their variants. PBMNCs from KIR typed healthy volunteers were stimulated with EBV-transformed target cells to enumerate NK cell response to EBV (degranulation and/or IFNγ production) as a function of KIR gene content and motifs' distribution using a multicolor flow cytometry-based assay. Effect of KIR gene profile on development of PTLD was analyzed using binomial competing risks regression statistics. Distribution of NK cell functional response across various KIR characterized groups was analyzed using Mann-Whitney U statistics.RESULTS:Donor telomeric A motifs (tA01, KIR3DL1+ve KIR2DS4+ve; KIR3DS1/2DS1+/-ve), strongly protected against PTLD (p=0.0001, SHR=0.17; Figure 1). An increased protection against PTLD with increasing number of tA01 was noted with at least one copy required for a significant protective effect (Figure 1B). Copy number analysis of tA01 gene contents yielded similar associations. Further, the number of EBV induced functional NK cell subsets were significantly higher in individuals with than without KIR genotypes containing tA01 motifs (Figure 2 A-C) and was found to be increasing with an increasing number of tA01 copies (Figure 2 A'-C'). There was no influence of recipients' KIR repertoire on the risk of developing PTLDCONCLUSIONS:NK cell responsiveness, a function of KIR gene repertoire has a profound effect on the development of PTLD. Appropriately characterized KIR gene profile based identification of HCT recipients at high risk of developing PTLD will enable closer monitoring of EBV DNAemia and facilitate prompt therapy. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Natural Killer Cell Functional Defects in Pediatric Patients With Severe and Recurrent Herpesvirus Infections

Natural killer (NK) cells play a critical role in the host defense against herpesviruses. Although herpesviruses are ubiquitous in human populations, only a minority of people experience severe recurrent infections. We hypothesize that uncharacterized NK cell functional deficits predispose individuals to more significant or frequent herpesvirus infections and reactivations. To investigate this hypothesis, we broadly analyzed NK cell phenotype and functional responses in a cohort of predominantly pediatric patients with recurrent and/or severe herpesvirus infections and compared them to a healthy control population. Our results identified no global differences in cytolysis, degranulation, interferon-γ production, or surface receptor upregulation following cytokine stimulation. However, abnormal NK cell functional responses were observed in nearly one-third of patients (including 3 with hyporesponsiveness to activating signals and 1 with markedly decreased CD11b expression associated with reduced cytotoxicity and degranulation), which might contribute to those individuals' susceptibility to herpesvirus infections.